biotinylated proteins Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Vector Laboratories biotinylated proteins
    Association of the FcRγ chain with the NKR-P1 molecule in IL-2–expanded NK cells. NK cells from normal mice expanded for 1 wk in the presence of IL-2 and were <t>surface-biotinylated.</t> The cell lysate of them was immunoprecipitated with anti-NK1.1 mAb ((Fab)′ 2 fragment) and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected as Fig. 1 A. FcRγ homodimer ( γ - γ ) was indicated.
    Biotinylated Proteins, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated proteins/product/Vector Laboratories
    Average 94 stars, based on 170 article reviews
    Price from $9.99 to $1999.99
    biotinylated proteins - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher avidin neutravidin biotin binding protein
    Autonomous PIP2 sorting in response to physical structures. a Schematic of the procedure of making PMSD pattern. b SEM images of PDMS micropatterns. c Biotinylated-GPMVs were labeled with BODIPY FL-PIP2, TopFluor-TMR PC, or PE and incubated with PDMS triangular and rectangular patterns coated with <t>NeutrAvidin.</t> Images were taken when GPMV were settled onto a specific pattern. Fold changes, defined as the ratio of fluorescence intensity of a given lipid at the pattern contacting regions over non-contacting regions, were measured for all shapes with or without 10 mM NaN 3 . n ≥ 30, N = 5. Scale bars, 5 µm. *** p
    Avidin Neutravidin Biotin Binding Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin neutravidin biotin binding protein/product/Thermo Fisher
    Average 99 stars, based on 200 article reviews
    Price from $9.99 to $1999.99
    avidin neutravidin biotin binding protein - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore biotinylated proteins
    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The <t>biotinylated</t> proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in
    Biotinylated Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated proteins/product/Millipore
    Average 99 stars, based on 1394 article reviews
    Price from $9.99 to $1999.99
    biotinylated proteins - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc biotinylated protein ladder
    fNKCC2AF: glycosylation, surface expression, and 86 Rb uptake. A , cell lysates from HEK-293 cells transfected with fNKCC2AF were incubated with (+) or without (−) PNGase F, subjected to SDS-PAGE, immunoblotted with anti-NKCC2 ( left panel ), and re-probed with N1 antibody ( right panel ). Molecular size markers (in kDa) and cross-reaction of anti-NKCC2 with NKCC1 (*) are indicated on the left and NKCC2AF is marked by an arrow on the right. B , 48 h after transfection with fNKCC2AF HEK-293 cells were surface <t>biotinylated</t> with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, run on SDS-PAGE, immunoblotted with anti-NKCC2 ( left ), and re-probed with N1 to detect endogenous NKCC1 ( right ). Twice as much protein was loaded and longer exposure times were used to detect surface proteins ( S ) compared with total proteins ( T ). C , untransfected ( HEK ) and fNKCC2AF-transfected ( AF ) HEK-293 cells, and D , stable fNKCC2A ( A ) and stable fNKCC2A cells co-expressing fNKCC2AF ( A+AF ) were grown to confluence (2–3 days). 86 Rb uptake was assessed following incubation of cells in isotonic (basal conditions, open bars ) or hypotonic/low-chloride ( filled bars ) medium. Bumetanide-sensitive 86 Rb uptakes are shown as mean ± S.E. Expression of fNKCC2AF has no significant ( n.s. ) effect on 86 Rb uptakes by HEK-293 cells ( C ) ( n ≥ 4) or HEK-293 cells stably expressing fNKCC2A ( D ) ( n ≥ 3).
    Biotinylated Protein Ladder, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 707 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein ladder/product/Cell Signaling Technology Inc
    Average 94 stars, based on 707 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein ladder - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    90
    Seikagaku biotinylated ha binding protein
    HA content of orthotopic tumor sections. Formalin-fixed, paraffin-embedded tumors were sectioned and stained with <t>biotinylated</t> HA binding protein, followed by detection with streptavidin-conjugated HRP and diaminobenzidine precipitation to visualize HA
    Biotinylated Ha Binding Protein, supplied by Seikagaku, used in various techniques. Bioz Stars score: 90/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated ha binding protein/product/Seikagaku
    Average 90 stars, based on 275 article reviews
    Price from $9.99 to $1999.99
    biotinylated ha binding protein - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    94
    Millipore biotinylated hyaluronic acid binding protein
    eIF4E overexpression correlates with increased HA synthesis. ( A ) Fluorescence staining of HA (in green) using <t>biotinylated</t> HA-binding protein with streptavidin-FITC in U2Os cells overexpressing eIF4E, S53A mutant or vector control in the presence or absence of Streptomyces Hyaluronidase treatment. DAPI is in blue. Note cell surface expression of HA in response to eIF4E overexpression. All confocal settings are identical between specimens and thus lower signal is indicative of less HA. A × 40 objective with no digital zoom was used. ( B ) 2x digital zoom in confocal images of HA from part ( A ). ( C ) Quantification of fluorophore-assisted carbohydrate electrophoresis (FACE) gels (Sup Figure 1e f ) for HA levels in U2Os cells expressing eIF4E, S53A mutant or vector control, and U2Os cells overexpressing eIF4E following HAS3/eIF4E knockdown or pharmacological inhibition with ribavirin (Rib). ( D ) Fluorescence staining of HA (in green) following siRNA to eIF4E or ribavirin treatment in U2Os cells overexpressing eIF4E. DAPI is in blue. A × 63 objective with no digital zoom used. For bar graphs, the mean ± SD are shown. Experiments were carried out in triplicate, at least three independent times. **p
    Biotinylated Hyaluronic Acid Binding Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated hyaluronic acid binding protein/product/Millipore
    Average 94 stars, based on 254 article reviews
    Price from $9.99 to $1999.99
    biotinylated hyaluronic acid binding protein - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    89
    Seikagaku biotinylated hyaluronic acid binding protein
    Effect of HA oligos on HA retention in MG-63 cells. MG-63 cells were treated with 250 μg/ml HA4 ( A ), HA8 ( B ), HMWHA ( C ), or control medium ( D ) for 72 hours, fixed in 2% paraformaldehyde, and then stained for HA using a <t>biotinylated</t> HABP reagent.
    Biotinylated Hyaluronic Acid Binding Protein, supplied by Seikagaku, used in various techniques. Bioz Stars score: 89/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated hyaluronic acid binding protein/product/Seikagaku
    Average 89 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    biotinylated hyaluronic acid binding protein - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    89
    Thermo Fisher biotinylated protein l
    The importance of linkage between the primary antibody and the magnetic particle for cell recovery. A, Schematic of different linkages tested: direct biotin-streptavidin linkage with <t>biotinylated</t> Fab (left panel), biotinylated secondary antibody binding non-biotinylated Fab (center panel), or biotinylated <t>Protein</t> L binding non-biotinylated Fab (right panel), in each case linked to the same streptavidin-magnetic particles. B, Efficiency of cell isolation using different linkages. BT474 were labeled with indicated concentrations of Fab0.11 and cell recoveries using the different linkages to the magnetic particle are shown (mean of triplicate ± 1 s.d.).
    Biotinylated Protein L, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein l/product/Thermo Fisher
    Average 89 stars, based on 161 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein l - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    90
    GenScript biotinylated protein l
    ( A ) Human primary T cells transduced with a CD19-CAR and parental cells were stained with purified CD19-ECD fusion protein labelled with Alexa Fluor- 647 (AF647). Alternatively, the cells were labeled with <t>Biotinylated</t> <t>Protein</t> L followed by APC-conjugated Streptavidin. After washes, cells were analyzed by flow cytometry. ( B ) Human primary T cells expressing a CD19-CAR or a CD33-CAR were incubated with 100 µl of supernatants containing the epitope-tagged CD19- or CD33-ECD fusion proteins for 45 minutes on ice. After incubation, cells were washed 3 times and incubated with the indicated anti-tag antibodies for 45 minutes. After 2 washes, cells were analyzed by flow cytometry. A representative of two independent experiments is shown.
    Biotinylated Protein L, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein l/product/GenScript
    Average 90 stars, based on 193 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein l - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    99
    Thermo Fisher cell surface protein biotinylation
    Subcellular localization of SR-BI/CLA-1 after the supply of postprandial micelles. (A) Immunoelectron micrograph of SR-BI/CLA-1 in untreated differentiated Caco-2/TC7 cells. MV, microvilli; TW, terminal web (bar, 0.5 µm). Note the significant amount of intracellular trafficking SR-BI/CLA-1 in addition to its main apical localization (arrowheads). (B) Immunolocalization of SR-BI/CLA-1 (green channel) and sucrase isomaltase (SI, red channel) in differentiated Caco-2/TC7 cells before (T0) and after 5, 10 and 15 min of apical PPM supply. Panels represent XY acquisitions at the apical level (bar, 10 µm). Arrowheads show clusters of SR-BI/CLA-1. (C) Immunolocalization of SR-BI/CLA-1 in differentiated Caco-2/TC7 cells in the absence (control) or presence of PPM or IPM for 20 min (bar, 20 µm). Arrowheads show clusters of SR-BI/CLA-1 (D) Immunoelectron micrograph of SR-BI/CLA-1 in Caco-2/TC7 cells supplied with PPM (MV, microvilli). Arrowheads indicate SR-BI/CLA-1 clusters (bar, 100 nm). (E) Cell surface <t>biotinylation</t> assay for apical SR-BI/CLA-1. Caco-2/TC7 cells were cultured in the absence (0) or presence of PPM for the indicated times. Cells were then selectively labeled with non-permeant biotin at the apical (left panel) or basal surface (right panel). Biotinylated fractions were obtained as described in Material and Methods . Total cell lysates (total), apical and basal biotinylated fractions (left and right panel respectively) and non-apical fractions (non-apical) were analyzed in immunoblots of SR-BI/CLA-1, E-cadherin being used as a basolateral membrane marker.
    Cell Surface Protein Biotinylation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell surface protein biotinylation/product/Thermo Fisher
    Average 99 stars, based on 275 article reviews
    Price from $9.99 to $1999.99
    cell surface protein biotinylation - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    97
    Thermo Fisher pierce far western blot kit for biotinylated proteins
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
    Pierce Far Western Blot Kit For Biotinylated Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pierce far western blot kit for biotinylated proteins/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pierce far western blot kit for biotinylated proteins - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    99
    GE Healthcare ecl protein biotinylation module
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
    Ecl Protein Biotinylation Module, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecl protein biotinylation module/product/GE Healthcare
    Average 99 stars, based on 150 article reviews
    Price from $9.99 to $1999.99
    ecl protein biotinylation module - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Vector Laboratories biotinylated protein a
    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. <t>Biotinylated</t> nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.
    Biotinylated Protein A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein a/product/Vector Laboratories
    Average 99 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Bio-Rad biotinylated protein
    Het α1 KO reduced β2 but not β3 subunit expression. A , we treated wild type brain slices with a membrane-impermeable biotinylation reagent and purified the <t>biotinylated</t> proteins with immobilized neutravidin. Equivalent fractions
    Biotinylated Protein, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein/product/Bio-Rad
    Average 91 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    94
    Thermo Fisher biotinylated protein a
    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using <t>biotinylated</t> <t>Protein</t> A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.
    Biotinylated Protein A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein a/product/Thermo Fisher
    Average 94 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein a - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    92
    Millipore biotinylated protein a
    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using <t>biotinylated</t> <t>Protein</t> A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.
    Biotinylated Protein A, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated protein a/product/Millipore
    Average 92 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    biotinylated protein a - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    91
    R&D Systems biotinylated ephrin a1 fc
    Relative EphA2 phosphorylation in the presence of different concentrations (50 μM, 25 μM, 12 μM, 6 μM) of compounds 1 (black), 2 (white) and 4 (sky blue). EphA2 phosphorylation was induced by treatment of PC3 cells with 0.25 μg/mL <t>ephrin-A1-Fc.</t> Cells were pretreated for 20 min with 1% DMSO or the indicated concentration of compounds and then stimulated for 20 min with ephrin-A1-Fc. Data are reported as a mean ± SEM of at least three independent experiments. One-way ANOVA followed by Dunnet’s post-test was performed to compare ephrin-A1-Fc + DMSO to all the other columns. * p
    Biotinylated Ephrin A1 Fc, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated ephrin a1 fc/product/R&D Systems
    Average 91 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    biotinylated ephrin a1 fc - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Association of the FcRγ chain with the NKR-P1 molecule in IL-2–expanded NK cells. NK cells from normal mice expanded for 1 wk in the presence of IL-2 and were surface-biotinylated. The cell lysate of them was immunoprecipitated with anti-NK1.1 mAb ((Fab)′ 2 fragment) and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected as Fig. 1 A. FcRγ homodimer ( γ - γ ) was indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: Association with FcR? Is Essential for Activation Signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T Cells

    doi:

    Figure Lengend Snippet: Association of the FcRγ chain with the NKR-P1 molecule in IL-2–expanded NK cells. NK cells from normal mice expanded for 1 wk in the presence of IL-2 and were surface-biotinylated. The cell lysate of them was immunoprecipitated with anti-NK1.1 mAb ((Fab)′ 2 fragment) and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected as Fig. 1 A. FcRγ homodimer ( γ - γ ) was indicated.

    Article Snippet: The biotinylated proteins were detected using streptavidin-peroxidase (VECSTAIN Elite ABC kit; Vector Laboratories Incorporated, Burlingame, CA), the ECL system ( Amersham International , Buckinghamshire, England), and autoradiography.

    Techniques: Mouse Assay, Immunoprecipitation, SDS Page

    Association of the FcRγ chain with the NKR-P1 molecule on the cell surface of CTLL-2 cell line. ( A ) CTLL-2 cells were surface-biotinylated and the cell lysate was immunoprecipitated with anti-NK1.1 mAb or anti-CD3ε mAb and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected by an ECL. ( B ) FcRγ chain was detected by blotting the membrane with anti-FcRγ Ab after immunoprecipitation with anti-NK1.1 or anti-CD3ε mAbs. FcRγ homodimer ( γ - γ ) and CD3ζ-FcRγ heterodimers ( ζ - γ ) were indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: Association with FcR? Is Essential for Activation Signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T Cells

    doi:

    Figure Lengend Snippet: Association of the FcRγ chain with the NKR-P1 molecule on the cell surface of CTLL-2 cell line. ( A ) CTLL-2 cells were surface-biotinylated and the cell lysate was immunoprecipitated with anti-NK1.1 mAb or anti-CD3ε mAb and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected by an ECL. ( B ) FcRγ chain was detected by blotting the membrane with anti-FcRγ Ab after immunoprecipitation with anti-NK1.1 or anti-CD3ε mAbs. FcRγ homodimer ( γ - γ ) and CD3ζ-FcRγ heterodimers ( ζ - γ ) were indicated.

    Article Snippet: The biotinylated proteins were detected using streptavidin-peroxidase (VECSTAIN Elite ABC kit; Vector Laboratories Incorporated, Burlingame, CA), the ECL system ( Amersham International , Buckinghamshire, England), and autoradiography.

    Techniques: Immunoprecipitation, SDS Page

    Autonomous PIP2 sorting in response to physical structures. a Schematic of the procedure of making PMSD pattern. b SEM images of PDMS micropatterns. c Biotinylated-GPMVs were labeled with BODIPY FL-PIP2, TopFluor-TMR PC, or PE and incubated with PDMS triangular and rectangular patterns coated with NeutrAvidin. Images were taken when GPMV were settled onto a specific pattern. Fold changes, defined as the ratio of fluorescence intensity of a given lipid at the pattern contacting regions over non-contacting regions, were measured for all shapes with or without 10 mM NaN 3 . n ≥ 30, N = 5. Scale bars, 5 µm. *** p

    Journal: Nature Communications

    Article Title: A phosphatidylinositol 4,5-bisphosphate redistribution-based sensing mechanism initiates a phagocytosis programing

    doi: 10.1038/s41467-018-06744-7

    Figure Lengend Snippet: Autonomous PIP2 sorting in response to physical structures. a Schematic of the procedure of making PMSD pattern. b SEM images of PDMS micropatterns. c Biotinylated-GPMVs were labeled with BODIPY FL-PIP2, TopFluor-TMR PC, or PE and incubated with PDMS triangular and rectangular patterns coated with NeutrAvidin. Images were taken when GPMV were settled onto a specific pattern. Fold changes, defined as the ratio of fluorescence intensity of a given lipid at the pattern contacting regions over non-contacting regions, were measured for all shapes with or without 10 mM NaN 3 . n ≥ 30, N = 5. Scale bars, 5 µm. *** p

    Article Snippet: To prepare for GPMV/pattern contact experiment, patterns were pre-coated with NeutrAvidin (A2666, Invitrogen) and stored at −20 °C.

    Techniques: Labeling, Incubation, Fluorescence

    J-protein promotes the interaction between Ssa1 and Hsp82 in the presence of ATP. (a) The in vitro interaction between biotinylated Hsp82 (2.6 μM) and Ssa1 (11 μM) alone and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. (b) The in vitro interaction between biotinylated Hsp82 (1.6 μM) and Ssa1 (8 μM) with Sti1 (1 μM) and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. In both (a) and (b) the results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 bound relative the amount bound in the absence of Ydj1 and ATP is plotted. The gels shown are representative of in (a) five and in (b) three independent experiments and quantification of the data from the replicate gels are presented as mean ± SEM.

    Journal: Journal of molecular biology

    Article Title: Intermolecular interactions between Hsp90 and Hsp70

    doi: 10.1016/j.jmb.2019.05.026

    Figure Lengend Snippet: J-protein promotes the interaction between Ssa1 and Hsp82 in the presence of ATP. (a) The in vitro interaction between biotinylated Hsp82 (2.6 μM) and Ssa1 (11 μM) alone and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. (b) The in vitro interaction between biotinylated Hsp82 (1.6 μM) and Ssa1 (8 μM) with Sti1 (1 μM) and in mixtures with increasing concentrations of Ydj1 in the absence or presence of 2 mM ATP was monitored using a pull-down assay as described in Materials and Methods. In both (a) and (b) the results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 bound relative the amount bound in the absence of Ydj1 and ATP is plotted. The gels shown are representative of in (a) five and in (b) three independent experiments and quantification of the data from the replicate gels are presented as mean ± SEM.

    Article Snippet: For each lane the biotinylated protein was used for normalization after any non-specific binding protein was subtracted.

    Techniques: In Vitro, Pull Down Assay, SDS Page, Staining, Binding Assay

    Specific crosslinking of a pair of Hsp90 Ec and DnaK residues important for interaction. (a) The in vivo interaction between Hsp90 Ec wild-type or K354C and DnaK wild-type or E209C was monitored using a bacterial two-hybrid system that measured beta-galactosidase activity in liquid culture. β-galactosidase activity is shown as mean ± SEM (n=3). (b) The in vitro interaction between biotinylated Hsp90 Ec wild-type or K354C and DnaK wild-type or E209C (4 μM each) was determined using a pull-down assay in the presence of L2, CbpA and ATP as described in Materials and Methods and analyzed by SDS-PAGE followed by Coomassie blue staining. DnaK wild-type or E209C associated with biotinylated Hsp90 Ec wild-type or K354C was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel the amount of DnaK was corrected for non-specific binding (NSB) and then was normalized to Hsp90 Ec -biotin and the ratio of DnaK mutant to wild-type is plotted as the mean ± SEM (n=6). (c) Hsp90 Ec -K354C and DnaK-E209C were treated with CuCl 2 (~2 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (*) indicates crosslinked product of interest. A small amount of a slowly migrating species was likely the covalently linked Hsp90 Ec -K354C dimer, since it was observed in reactions with and without Ssa1 (lanes 3 and 4). In (c), the gel shown is representative of at least three independent experiments.

    Journal: Journal of molecular biology

    Article Title: Intermolecular interactions between Hsp90 and Hsp70

    doi: 10.1016/j.jmb.2019.05.026

    Figure Lengend Snippet: Specific crosslinking of a pair of Hsp90 Ec and DnaK residues important for interaction. (a) The in vivo interaction between Hsp90 Ec wild-type or K354C and DnaK wild-type or E209C was monitored using a bacterial two-hybrid system that measured beta-galactosidase activity in liquid culture. β-galactosidase activity is shown as mean ± SEM (n=3). (b) The in vitro interaction between biotinylated Hsp90 Ec wild-type or K354C and DnaK wild-type or E209C (4 μM each) was determined using a pull-down assay in the presence of L2, CbpA and ATP as described in Materials and Methods and analyzed by SDS-PAGE followed by Coomassie blue staining. DnaK wild-type or E209C associated with biotinylated Hsp90 Ec wild-type or K354C was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel the amount of DnaK was corrected for non-specific binding (NSB) and then was normalized to Hsp90 Ec -biotin and the ratio of DnaK mutant to wild-type is plotted as the mean ± SEM (n=6). (c) Hsp90 Ec -K354C and DnaK-E209C were treated with CuCl 2 (~2 Å linker) alone and in a mixture together and covalent bond formation was monitored by SDS-PAGE followed by Coomassie blue staining. (*) indicates crosslinked product of interest. A small amount of a slowly migrating species was likely the covalently linked Hsp90 Ec -K354C dimer, since it was observed in reactions with and without Ssa1 (lanes 3 and 4). In (c), the gel shown is representative of at least three independent experiments.

    Article Snippet: For each lane the biotinylated protein was used for normalization after any non-specific binding protein was subtracted.

    Techniques: In Vivo, Activity Assay, In Vitro, Pull Down Assay, SDS Page, Staining, Binding Assay, Mutagenesis

    Ssa1 residues homologous to DnaK residues important for interacting with Hsp90 Ec are important for Hsp82 and Ydj1 binding. (a) Chart showing residues in S. cerevisiae Hsp70, Ssa1, that align with four residues in E. coli DnaK, that were previously shown to be in the Hsp90 Ec ). Residues mutated in this study and tested for interaction with Hsp82 are shown in color and labeled. (c) The in vitro interaction between biotinylated Hsp82 (2.5 μM) and Ssa1 wild-type or mutant (8 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (d) The in vitro interaction between biotinylated Ydj1 (1.2 μM) with Ssa1 wild-type or mutant (2 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Ydj1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Ydj1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (e) Heat-inactivated luciferase reactivation by Ssa1 wild-type or mutant and Ydj1 as indicated and described in Materials and Methods. Data from three or more replicates are presented as mean ± SEM. For some points, the symbols obscure the error bars. (f) The in vitro interaction between biotinylated Sti1 (2 μM) and Ssa1 wild-type or mutant (4 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Sti1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Sti1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. In (c, d and f), the gels shown are representative of at least three independent experiments and quantification of the data from three or more replicate gels are presented as mean ± SEM.

    Journal: Journal of molecular biology

    Article Title: Intermolecular interactions between Hsp90 and Hsp70

    doi: 10.1016/j.jmb.2019.05.026

    Figure Lengend Snippet: Ssa1 residues homologous to DnaK residues important for interacting with Hsp90 Ec are important for Hsp82 and Ydj1 binding. (a) Chart showing residues in S. cerevisiae Hsp70, Ssa1, that align with four residues in E. coli DnaK, that were previously shown to be in the Hsp90 Ec ). Residues mutated in this study and tested for interaction with Hsp82 are shown in color and labeled. (c) The in vitro interaction between biotinylated Hsp82 (2.5 μM) and Ssa1 wild-type or mutant (8 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Hsp82 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Hsp82-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (d) The in vitro interaction between biotinylated Ydj1 (1.2 μM) with Ssa1 wild-type or mutant (2 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Ydj1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Ydj1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. (e) Heat-inactivated luciferase reactivation by Ssa1 wild-type or mutant and Ydj1 as indicated and described in Materials and Methods. Data from three or more replicates are presented as mean ± SEM. For some points, the symbols obscure the error bars. (f) The in vitro interaction between biotinylated Sti1 (2 μM) and Ssa1 wild-type or mutant (4 μM) was monitored using a pull-down assay as described in Materials and Methods. The results were analyzed by SDS-PAGE followed by Coomassie blue staining. Ssa1 wild-type or mutant associated with biotinylated Sti1 was quantified using densitometry as described in Materials and Methods and is shown as a bar graph. For each lane of the gel, the amount of Ssa1 was corrected for non-specific binding (NSB) and then was normalized to Sti1-biotin and the ratio of Ssa1 mutant to wild-type is plotted. In (c, d and f), the gels shown are representative of at least three independent experiments and quantification of the data from three or more replicate gels are presented as mean ± SEM.

    Article Snippet: For each lane the biotinylated protein was used for normalization after any non-specific binding protein was subtracted.

    Techniques: Binding Assay, Labeling, In Vitro, Mutagenesis, Pull Down Assay, SDS Page, Staining, Luciferase

    Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The biotinylated proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in

    Journal: Molecular Pain

    Article Title: Involvement of S-nitrosylation of actin in inhibition of neurotransmitter release by nitric oxide

    doi: 10.1186/1744-8069-5-58

    Figure Lengend Snippet: Identification of protein S -nitrosylated by SNAP in the spinal cord as actin . A . S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S -nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B . The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The biotinylated proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C . MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D . Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E . S -Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S -nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in "Methods."

    Article Snippet: Biotinylated proteins were resolved by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride membrane, followed by immunoblotting with peroxidase-conjugated anti-biotin antibody (1:1000; Sigma-Aldrich).

    Techniques: Incubation, Biotin Switch Assay, SDS Page, Purification, Agarose Gel Electrophoresis, Silver Staining, Mass Spectrometry, Sequencing

    Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .

    Journal: PLoS ONE

    Article Title: Tumor-Specific Hsp70 Plasma Membrane Localization Is Enabled by the Glycosphingolipid Gb3

    doi: 10.1371/journal.pone.0001925

    Figure Lengend Snippet: Quantification of cytosolic and plasma membrane-bound Hsp70 in CX+/CX− and Colo+/Colo− carcinoma sublines. Western blot analysis of biotinylated whole cell lysates (cytoplasm, upper graph), and plasma membranes (lower graph) of CX+/CX− and Colo+/Colo− tumor sublines. A corresponding Western blot was stained with the Hsp70 specific mAb cmHsp70.1. The figure shows a representative blot from three independent experiments. The Hsp70 surface phenotypes of the tumor sublines, as determined by flow cytometric analysis, are summarized in Table 1 .

    Article Snippet: Biotinylated membrane proteins were eluted from the column with 5 mM biotin in PBS containing 1% v/v Triton-X, concentrated on Centricon YM-3 columns (Millipore) and subjected to SDS-PAGE.

    Techniques: Western Blot, Staining, Flow Cytometry

    fNKCC2AF: glycosylation, surface expression, and 86 Rb uptake. A , cell lysates from HEK-293 cells transfected with fNKCC2AF were incubated with (+) or without (−) PNGase F, subjected to SDS-PAGE, immunoblotted with anti-NKCC2 ( left panel ), and re-probed with N1 antibody ( right panel ). Molecular size markers (in kDa) and cross-reaction of anti-NKCC2 with NKCC1 (*) are indicated on the left and NKCC2AF is marked by an arrow on the right. B , 48 h after transfection with fNKCC2AF HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, run on SDS-PAGE, immunoblotted with anti-NKCC2 ( left ), and re-probed with N1 to detect endogenous NKCC1 ( right ). Twice as much protein was loaded and longer exposure times were used to detect surface proteins ( S ) compared with total proteins ( T ). C , untransfected ( HEK ) and fNKCC2AF-transfected ( AF ) HEK-293 cells, and D , stable fNKCC2A ( A ) and stable fNKCC2A cells co-expressing fNKCC2AF ( A+AF ) were grown to confluence (2–3 days). 86 Rb uptake was assessed following incubation of cells in isotonic (basal conditions, open bars ) or hypotonic/low-chloride ( filled bars ) medium. Bumetanide-sensitive 86 Rb uptakes are shown as mean ± S.E. Expression of fNKCC2AF has no significant ( n.s. ) effect on 86 Rb uptakes by HEK-293 cells ( C ) ( n ≥ 4) or HEK-293 cells stably expressing fNKCC2A ( D ) ( n ≥ 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant *

    doi: 10.1074/jbc.M109.060004

    Figure Lengend Snippet: fNKCC2AF: glycosylation, surface expression, and 86 Rb uptake. A , cell lysates from HEK-293 cells transfected with fNKCC2AF were incubated with (+) or without (−) PNGase F, subjected to SDS-PAGE, immunoblotted with anti-NKCC2 ( left panel ), and re-probed with N1 antibody ( right panel ). Molecular size markers (in kDa) and cross-reaction of anti-NKCC2 with NKCC1 (*) are indicated on the left and NKCC2AF is marked by an arrow on the right. B , 48 h after transfection with fNKCC2AF HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, run on SDS-PAGE, immunoblotted with anti-NKCC2 ( left ), and re-probed with N1 to detect endogenous NKCC1 ( right ). Twice as much protein was loaded and longer exposure times were used to detect surface proteins ( S ) compared with total proteins ( T ). C , untransfected ( HEK ) and fNKCC2AF-transfected ( AF ) HEK-293 cells, and D , stable fNKCC2A ( A ) and stable fNKCC2A cells co-expressing fNKCC2AF ( A+AF ) were grown to confluence (2–3 days). 86 Rb uptake was assessed following incubation of cells in isotonic (basal conditions, open bars ) or hypotonic/low-chloride ( filled bars ) medium. Bumetanide-sensitive 86 Rb uptakes are shown as mean ± S.E. Expression of fNKCC2AF has no significant ( n.s. ) effect on 86 Rb uptakes by HEK-293 cells ( C ) ( n ≥ 4) or HEK-293 cells stably expressing fNKCC2A ( D ) ( n ≥ 3).

    Article Snippet: Biotinylated protein ladder (Cell Signaling Technology, Danvers, MA) and MagicMark XP marker (Invitrogen) were used for estimation of molecular weights.

    Techniques: Expressing, Transfection, Incubation, SDS Page, Stable Transfection

    fNKCC2 glycosylation and appearance in the cell surface. A , whole cell lysates from HEK-293 cells transfected with fNKCC2A, -B, and -F were treated with (+) or without (−) PNGase F, subjected to SDS-PAGE, and immunoblotted with anti-NKCC2 ( left panel ) and re-probed with T4 ( right panel ). Exposure time was increased to detect fNKCC2F (shown by vertical line ). B , detection of surface proteins. 48 h after transfection HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, eluted, run on SDS-PAGE, and immunoblotted with anti-NKCC2 ( left panels ) and re-probed with N1 to detect endogenous NKCC1 ( right panels ). Twice as much protein was loaded and longer exposure times were used to detect surface proteins compared with total proteins. Molecular mass markers (in kDa) are indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant *

    doi: 10.1074/jbc.M109.060004

    Figure Lengend Snippet: fNKCC2 glycosylation and appearance in the cell surface. A , whole cell lysates from HEK-293 cells transfected with fNKCC2A, -B, and -F were treated with (+) or without (−) PNGase F, subjected to SDS-PAGE, and immunoblotted with anti-NKCC2 ( left panel ) and re-probed with T4 ( right panel ). Exposure time was increased to detect fNKCC2F (shown by vertical line ). B , detection of surface proteins. 48 h after transfection HEK-293 cells were surface biotinylated with Sulfo-NHS-LC-biotin. Biotinylated proteins were precipitated from cell lysates with streptavidin beads, eluted, run on SDS-PAGE, and immunoblotted with anti-NKCC2 ( left panels ) and re-probed with N1 to detect endogenous NKCC1 ( right panels ). Twice as much protein was loaded and longer exposure times were used to detect surface proteins compared with total proteins. Molecular mass markers (in kDa) are indicated.

    Article Snippet: Biotinylated protein ladder (Cell Signaling Technology, Danvers, MA) and MagicMark XP marker (Invitrogen) were used for estimation of molecular weights.

    Techniques: Transfection, SDS Page

    HA content of orthotopic tumor sections. Formalin-fixed, paraffin-embedded tumors were sectioned and stained with biotinylated HA binding protein, followed by detection with streptavidin-conjugated HRP and diaminobenzidine precipitation to visualize HA

    Journal:

    Article Title: Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

    doi: 10.2353/ajpath.2009.080501

    Figure Lengend Snippet: HA content of orthotopic tumor sections. Formalin-fixed, paraffin-embedded tumors were sectioned and stained with biotinylated HA binding protein, followed by detection with streptavidin-conjugated HRP and diaminobenzidine precipitation to visualize HA

    Article Snippet: Biotinylated HA binding protein was from Seikagaku (Tokyo, Japan).

    Techniques: Formalin-fixed Paraffin-Embedded, Staining, Binding Assay

    eIF4E overexpression correlates with increased HA synthesis. ( A ) Fluorescence staining of HA (in green) using biotinylated HA-binding protein with streptavidin-FITC in U2Os cells overexpressing eIF4E, S53A mutant or vector control in the presence or absence of Streptomyces Hyaluronidase treatment. DAPI is in blue. Note cell surface expression of HA in response to eIF4E overexpression. All confocal settings are identical between specimens and thus lower signal is indicative of less HA. A × 40 objective with no digital zoom was used. ( B ) 2x digital zoom in confocal images of HA from part ( A ). ( C ) Quantification of fluorophore-assisted carbohydrate electrophoresis (FACE) gels (Sup Figure 1e f ) for HA levels in U2Os cells expressing eIF4E, S53A mutant or vector control, and U2Os cells overexpressing eIF4E following HAS3/eIF4E knockdown or pharmacological inhibition with ribavirin (Rib). ( D ) Fluorescence staining of HA (in green) following siRNA to eIF4E or ribavirin treatment in U2Os cells overexpressing eIF4E. DAPI is in blue. A × 63 objective with no digital zoom used. For bar graphs, the mean ± SD are shown. Experiments were carried out in triplicate, at least three independent times. **p

    Journal: eLife

    Article Title: The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity

    doi: 10.7554/eLife.29830

    Figure Lengend Snippet: eIF4E overexpression correlates with increased HA synthesis. ( A ) Fluorescence staining of HA (in green) using biotinylated HA-binding protein with streptavidin-FITC in U2Os cells overexpressing eIF4E, S53A mutant or vector control in the presence or absence of Streptomyces Hyaluronidase treatment. DAPI is in blue. Note cell surface expression of HA in response to eIF4E overexpression. All confocal settings are identical between specimens and thus lower signal is indicative of less HA. A × 40 objective with no digital zoom was used. ( B ) 2x digital zoom in confocal images of HA from part ( A ). ( C ) Quantification of fluorophore-assisted carbohydrate electrophoresis (FACE) gels (Sup Figure 1e f ) for HA levels in U2Os cells expressing eIF4E, S53A mutant or vector control, and U2Os cells overexpressing eIF4E following HAS3/eIF4E knockdown or pharmacological inhibition with ribavirin (Rib). ( D ) Fluorescence staining of HA (in green) following siRNA to eIF4E or ribavirin treatment in U2Os cells overexpressing eIF4E. DAPI is in blue. A × 63 objective with no digital zoom used. For bar graphs, the mean ± SD are shown. Experiments were carried out in triplicate, at least three independent times. **p

    Article Snippet: Immunofluorescence, fluorescence and laser-scanning confocal microscopy For HA staining, biotinylated hyaluronic acid binding protein (from Bovine Nasal Cartilage, Millipore, Cat# 385911) resuspended in 100 µl water: glycerol (50:50) was used.

    Techniques: Over Expression, Fluorescence, Staining, Binding Assay, Mutagenesis, Plasmid Preparation, Expressing, Electrophoresis, Inhibition

    HA biosynthesis is required for eIF4E-mediated lung metastasis in mice. ( A ) Histochemical staining of HA using biotinylated HABP in metastatic mouse tumors. +HAse indicates sections were treated with HAse prior to biotinylated HABP to ensure HA staining was specific. Red arrows indicate tumors. 3X (first and third row) and 50X (second row) magnification are presented. ( B ) Quantification from Visiomorph for all 19 mice over multiple sections per animal. For bar graphs, the mean ± standard deviation are shown. *p

    Journal: eLife

    Article Title: The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity

    doi: 10.7554/eLife.29830

    Figure Lengend Snippet: HA biosynthesis is required for eIF4E-mediated lung metastasis in mice. ( A ) Histochemical staining of HA using biotinylated HABP in metastatic mouse tumors. +HAse indicates sections were treated with HAse prior to biotinylated HABP to ensure HA staining was specific. Red arrows indicate tumors. 3X (first and third row) and 50X (second row) magnification are presented. ( B ) Quantification from Visiomorph for all 19 mice over multiple sections per animal. For bar graphs, the mean ± standard deviation are shown. *p

    Article Snippet: Immunofluorescence, fluorescence and laser-scanning confocal microscopy For HA staining, biotinylated hyaluronic acid binding protein (from Bovine Nasal Cartilage, Millipore, Cat# 385911) resuspended in 100 µl water: glycerol (50:50) was used.

    Techniques: Mouse Assay, Staining, Standard Deviation

    Effect of HA oligos on HA retention in MG-63 cells. MG-63 cells were treated with 250 μg/ml HA4 ( A ), HA8 ( B ), HMWHA ( C ), or control medium ( D ) for 72 hours, fixed in 2% paraformaldehyde, and then stained for HA using a biotinylated HABP reagent.

    Journal:

    Article Title: Hyaluronan Oligosaccharides Inhibit Tumorigenicity of Osteosarcoma Cell Lines MG-63 and LM-8 in Vitro and in Vivo via Perturbation of Hyaluronan-Rich Pericellular Matrix of the Cells

    doi: 10.2353/ajpath.2007.060828

    Figure Lengend Snippet: Effect of HA oligos on HA retention in MG-63 cells. MG-63 cells were treated with 250 μg/ml HA4 ( A ), HA8 ( B ), HMWHA ( C ), or control medium ( D ) for 72 hours, fixed in 2% paraformaldehyde, and then stained for HA using a biotinylated HABP reagent.

    Article Snippet: Biotinylated hyaluronic acid binding protein was purchased from Seikagaku America (Falmouth, MA).

    Techniques: Staining

    The importance of linkage between the primary antibody and the magnetic particle for cell recovery. A, Schematic of different linkages tested: direct biotin-streptavidin linkage with biotinylated Fab (left panel), biotinylated secondary antibody binding non-biotinylated Fab (center panel), or biotinylated Protein L binding non-biotinylated Fab (right panel), in each case linked to the same streptavidin-magnetic particles. B, Efficiency of cell isolation using different linkages. BT474 were labeled with indicated concentrations of Fab0.11 and cell recoveries using the different linkages to the magnetic particle are shown (mean of triplicate ± 1 s.d.).

    Journal: Cancer research

    Article Title: Cholesterol loading and ultrastable protein interactions determine the level of tumor marker required for optimal isolation of cancer cells

    doi: 10.1158/0008-5472.CAN-12-2956

    Figure Lengend Snippet: The importance of linkage between the primary antibody and the magnetic particle for cell recovery. A, Schematic of different linkages tested: direct biotin-streptavidin linkage with biotinylated Fab (left panel), biotinylated secondary antibody binding non-biotinylated Fab (center panel), or biotinylated Protein L binding non-biotinylated Fab (right panel), in each case linked to the same streptavidin-magnetic particles. B, Efficiency of cell isolation using different linkages. BT474 were labeled with indicated concentrations of Fab0.11 and cell recoveries using the different linkages to the magnetic particle are shown (mean of triplicate ± 1 s.d.).

    Article Snippet: For testing different linkages to the anti-HER2 primary antibody, 25 μl Dynabeads® Biotin Binder were coated with 3 μg biotinylated goat anti-human kappa chain (Thermo Scientific) or 3 μg biotinylated Protein L (Thermo Scientific) in 10 μL PBS for 10 min at 25°C before washing twice with PBS and once with D1.

    Techniques: Binding Assay, Cell Isolation, Labeling

    Close proximity of cadherin molecules in a cis orientation cooperatively increases the probability of trans cadherin binding. ( A ) Schematic of AFM tip and substrate functionalized with cadherin–Fc dimers for single-molecule force measurements. The tip and surface were functionalized with PEG linkers, some of which were decorated with streptavidin molecules. Cadherin–Fc dimers were immobilized on biotinylated protein G attached to the streptavidin molecules on the tip and surface. The flexible PEG linkers enable unhindered interactions between opposing cadherins during tip–substrate encounters. Cadherin–Fc dimers were immobilized on the AFM tip and substrate at a surface density of 34 ± 16 dimers per μm 2 . ( B ) Schematic of AFM tip and substrate functionalized with biotinylated cadherin monomers for single-molecule force measurements. The biotinylated cadherin monomers were immobilized on an AFM tip and substrate via streptavidin-PEG tethers at a surface densities of 65 ± 18 monomers per μm 2 ; thus, the protein surface density for the cadherin monomers and cadherin–Fc dimer constructs are similar. ( C ) A typical force curve showing the unbinding of a single cadherin molecule. The tip and the substrate decorated with cadherins were bought into contact for either 1,100, 340, or 115 ms so that cadherins on the tip and substrate formed an adhesive complex. When the tip was withdrawn from the substrate a force was exerted, and above a critical force the adhesive complex ruptured. Forces between the cadherin–Fc dimers were measured 7,995 times in 2.5 mM Ca 2+ , yielding 509 binding events, and 5,931 times in 2 mM EGTA, yielding 123 binding events. Forces between the cadherin monomers were measured 5,949 times in 2.5 mM Ca 2+ , yielding 113 binding events, and 5,846 times in 2 mM EGTA, yielding 28 binding events. ( D ) Histogram of cadherin–Fc dimer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 53 ± 27 pN. The binding probability of cadherin–Fc dimers at contact times of 1,100, 340, or 115 ms was 8.6%, 6.6%, and 3.7%, respectively. The histogram includes the forces measured at all 3 tip-surface contact times. ( E ) Histogram of cadherin monomer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The histogram includes the forces measured at all 3 tip-surface contact times (see above). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 64 ± 27 pN. Although the bond strengths of the monomer and cadherin–Fc dimer adhesive complexes are similar, the cadherin–Fc dimer has a higher probability of binding than a cadherin monomer. The total probability of cadherin monomer binding at contact times of 1,100, 340, or 115 ms was 2.5%, 2.2%, and 1.1% respectively. Thus, the cadherin–Fc dimer binding probability was 3.5, 3.1, and 3.6 times higher than the probability of monomer interactions at these tip-surface contact times.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Resolving cadherin interactions and binding cooperativity at the single-molecule level

    doi: 10.1073/pnas.0811350106

    Figure Lengend Snippet: Close proximity of cadherin molecules in a cis orientation cooperatively increases the probability of trans cadherin binding. ( A ) Schematic of AFM tip and substrate functionalized with cadherin–Fc dimers for single-molecule force measurements. The tip and surface were functionalized with PEG linkers, some of which were decorated with streptavidin molecules. Cadherin–Fc dimers were immobilized on biotinylated protein G attached to the streptavidin molecules on the tip and surface. The flexible PEG linkers enable unhindered interactions between opposing cadherins during tip–substrate encounters. Cadherin–Fc dimers were immobilized on the AFM tip and substrate at a surface density of 34 ± 16 dimers per μm 2 . ( B ) Schematic of AFM tip and substrate functionalized with biotinylated cadherin monomers for single-molecule force measurements. The biotinylated cadherin monomers were immobilized on an AFM tip and substrate via streptavidin-PEG tethers at a surface densities of 65 ± 18 monomers per μm 2 ; thus, the protein surface density for the cadherin monomers and cadherin–Fc dimer constructs are similar. ( C ) A typical force curve showing the unbinding of a single cadherin molecule. The tip and the substrate decorated with cadherins were bought into contact for either 1,100, 340, or 115 ms so that cadherins on the tip and substrate formed an adhesive complex. When the tip was withdrawn from the substrate a force was exerted, and above a critical force the adhesive complex ruptured. Forces between the cadherin–Fc dimers were measured 7,995 times in 2.5 mM Ca 2+ , yielding 509 binding events, and 5,931 times in 2 mM EGTA, yielding 123 binding events. Forces between the cadherin monomers were measured 5,949 times in 2.5 mM Ca 2+ , yielding 113 binding events, and 5,846 times in 2 mM EGTA, yielding 28 binding events. ( D ) Histogram of cadherin–Fc dimer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 53 ± 27 pN. The binding probability of cadherin–Fc dimers at contact times of 1,100, 340, or 115 ms was 8.6%, 6.6%, and 3.7%, respectively. The histogram includes the forces measured at all 3 tip-surface contact times. ( E ) Histogram of cadherin monomer binding events measured in 2.5 mM Ca 2+ (solid gray bars) and 2 mM EGTA (hatched green bars). The histogram includes the forces measured at all 3 tip-surface contact times (see above). The binding events measured in 2.5 mM Ca 2+ were fitted to a Gaussian distribution with a peak force of 64 ± 27 pN. Although the bond strengths of the monomer and cadherin–Fc dimer adhesive complexes are similar, the cadherin–Fc dimer has a higher probability of binding than a cadherin monomer. The total probability of cadherin monomer binding at contact times of 1,100, 340, or 115 ms was 2.5%, 2.2%, and 1.1% respectively. Thus, the cadherin–Fc dimer binding probability was 3.5, 3.1, and 3.6 times higher than the probability of monomer interactions at these tip-surface contact times.

    Article Snippet: To immobilize Fc–cadherin dimers, the streptavidins were decorated with biotinylated protein G (Pierce Biotechnology), and the Fc region of the cadherin dimer was specifically bound to the protein G. Fluorescently-labeled cadherin molecules were observed by using a home-built prism-type total internal reflection fluorescence microscope.

    Techniques: Binding Assay, Construct, Mass Spectrometry

    Fig. 4. ( A ) Western blot detection of cell surface-bound E2. Biotinylated HepG2 cells were incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of E2 recombinant protein. Bound E2 was cross-linked with DTSSP and the E2–receptor complexes were immunoprecipitated with an antibody against the His tag of the E2 recombinant protein. Samples eluted under both non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions were loaded onto a 10% SDS–PAGE gel. E2 protein was detected as a monomer under reducing conditions (lane 3) and at a higher molecular weight under non- reducing conditions (lane 1), by using an anti-E2 rat mAb followed by anti-rat HRP-conjugated. ( B ) Immunoblot detection of biotinylated cell surface proteins interacting with E2. The reactivity with HRP conjugated streptavidin revealed a biotinylated protein band at 82 kDa only under reducing conditions (lane 3).

    Journal: The EMBO Journal

    Article Title: The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

    doi: 10.1093/emboj/cdf529

    Figure Lengend Snippet: Fig. 4. ( A ) Western blot detection of cell surface-bound E2. Biotinylated HepG2 cells were incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4) of E2 recombinant protein. Bound E2 was cross-linked with DTSSP and the E2–receptor complexes were immunoprecipitated with an antibody against the His tag of the E2 recombinant protein. Samples eluted under both non-reducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions were loaded onto a 10% SDS–PAGE gel. E2 protein was detected as a monomer under reducing conditions (lane 3) and at a higher molecular weight under non- reducing conditions (lane 1), by using an anti-E2 rat mAb followed by anti-rat HRP-conjugated. ( B ) Immunoblot detection of biotinylated cell surface proteins interacting with E2. The reactivity with HRP conjugated streptavidin revealed a biotinylated protein band at 82 kDa only under reducing conditions (lane 3).

    Article Snippet: Samples obtained by boiling the beads were loaded on to an SDS–PAGE gel and analyzed by western blot for detection of biotinylated proteins using horseradish peroxidase (HRP)-conjugated streptavidin (Pierce) diluted 1:25 000 in Tris-buffered saline 0.05% Tween 20 (TBST), 2% bovine serum albumin (BSA).

    Techniques: Western Blot, Incubation, Recombinant, Immunoprecipitation, SDS Page, Molecular Weight

    ( A ) Human primary T cells transduced with a CD19-CAR and parental cells were stained with purified CD19-ECD fusion protein labelled with Alexa Fluor- 647 (AF647). Alternatively, the cells were labeled with Biotinylated Protein L followed by APC-conjugated Streptavidin. After washes, cells were analyzed by flow cytometry. ( B ) Human primary T cells expressing a CD19-CAR or a CD33-CAR were incubated with 100 µl of supernatants containing the epitope-tagged CD19- or CD33-ECD fusion proteins for 45 minutes on ice. After incubation, cells were washed 3 times and incubated with the indicated anti-tag antibodies for 45 minutes. After 2 washes, cells were analyzed by flow cytometry. A representative of two independent experiments is shown.

    Journal: Scientific Reports

    Article Title: A novel luciferase-based assay for the detection of Chimeric Antigen Receptors

    doi: 10.1038/s41598-018-38258-z

    Figure Lengend Snippet: ( A ) Human primary T cells transduced with a CD19-CAR and parental cells were stained with purified CD19-ECD fusion protein labelled with Alexa Fluor- 647 (AF647). Alternatively, the cells were labeled with Biotinylated Protein L followed by APC-conjugated Streptavidin. After washes, cells were analyzed by flow cytometry. ( B ) Human primary T cells expressing a CD19-CAR or a CD33-CAR were incubated with 100 µl of supernatants containing the epitope-tagged CD19- or CD33-ECD fusion proteins for 45 minutes on ice. After incubation, cells were washed 3 times and incubated with the indicated anti-tag antibodies for 45 minutes. After 2 washes, cells were analyzed by flow cytometry. A representative of two independent experiments is shown.

    Article Snippet: Strep-Tactin-APC (IBA-lifescience: Cat# 6-5010-001); Anti-Flag-FITC (Sigma: F4049); Anti-Strep-tagII-FITC (Genscript: A01736); Biotinylated Protein L (Genscript: M0097); Streptavidin-APC conjugate (Molecular probes: SA1005); APC-conjugated anti-MYC antibody (R & D systems: IC3696A); and Polyethylenimine (Polysciences Inc: 23966) were obtained from the indicated sources.

    Techniques: Transduction, Staining, Purification, Labeling, Flow Cytometry, Cytometry, Expressing, Incubation

    Subcellular localization of SR-BI/CLA-1 after the supply of postprandial micelles. (A) Immunoelectron micrograph of SR-BI/CLA-1 in untreated differentiated Caco-2/TC7 cells. MV, microvilli; TW, terminal web (bar, 0.5 µm). Note the significant amount of intracellular trafficking SR-BI/CLA-1 in addition to its main apical localization (arrowheads). (B) Immunolocalization of SR-BI/CLA-1 (green channel) and sucrase isomaltase (SI, red channel) in differentiated Caco-2/TC7 cells before (T0) and after 5, 10 and 15 min of apical PPM supply. Panels represent XY acquisitions at the apical level (bar, 10 µm). Arrowheads show clusters of SR-BI/CLA-1. (C) Immunolocalization of SR-BI/CLA-1 in differentiated Caco-2/TC7 cells in the absence (control) or presence of PPM or IPM for 20 min (bar, 20 µm). Arrowheads show clusters of SR-BI/CLA-1 (D) Immunoelectron micrograph of SR-BI/CLA-1 in Caco-2/TC7 cells supplied with PPM (MV, microvilli). Arrowheads indicate SR-BI/CLA-1 clusters (bar, 100 nm). (E) Cell surface biotinylation assay for apical SR-BI/CLA-1. Caco-2/TC7 cells were cultured in the absence (0) or presence of PPM for the indicated times. Cells were then selectively labeled with non-permeant biotin at the apical (left panel) or basal surface (right panel). Biotinylated fractions were obtained as described in Material and Methods . Total cell lysates (total), apical and basal biotinylated fractions (left and right panel respectively) and non-apical fractions (non-apical) were analyzed in immunoblots of SR-BI/CLA-1, E-cadherin being used as a basolateral membrane marker.

    Journal: PLoS ONE

    Article Title: Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

    doi: 10.1371/journal.pone.0004278

    Figure Lengend Snippet: Subcellular localization of SR-BI/CLA-1 after the supply of postprandial micelles. (A) Immunoelectron micrograph of SR-BI/CLA-1 in untreated differentiated Caco-2/TC7 cells. MV, microvilli; TW, terminal web (bar, 0.5 µm). Note the significant amount of intracellular trafficking SR-BI/CLA-1 in addition to its main apical localization (arrowheads). (B) Immunolocalization of SR-BI/CLA-1 (green channel) and sucrase isomaltase (SI, red channel) in differentiated Caco-2/TC7 cells before (T0) and after 5, 10 and 15 min of apical PPM supply. Panels represent XY acquisitions at the apical level (bar, 10 µm). Arrowheads show clusters of SR-BI/CLA-1. (C) Immunolocalization of SR-BI/CLA-1 in differentiated Caco-2/TC7 cells in the absence (control) or presence of PPM or IPM for 20 min (bar, 20 µm). Arrowheads show clusters of SR-BI/CLA-1 (D) Immunoelectron micrograph of SR-BI/CLA-1 in Caco-2/TC7 cells supplied with PPM (MV, microvilli). Arrowheads indicate SR-BI/CLA-1 clusters (bar, 100 nm). (E) Cell surface biotinylation assay for apical SR-BI/CLA-1. Caco-2/TC7 cells were cultured in the absence (0) or presence of PPM for the indicated times. Cells were then selectively labeled with non-permeant biotin at the apical (left panel) or basal surface (right panel). Biotinylated fractions were obtained as described in Material and Methods . Total cell lysates (total), apical and basal biotinylated fractions (left and right panel respectively) and non-apical fractions (non-apical) were analyzed in immunoblots of SR-BI/CLA-1, E-cadherin being used as a basolateral membrane marker.

    Article Snippet: Apical membrane expression of SR-BI/CLA-1 was analyzed by biotin labeling of surface proteins with EZ-Link Sulfo-NHS-SS-Biotin, followed by immunoprecipitation with immobilized streptavidin using the Cell Surface Protein Biotinylation and Purification Kit (Pierce), according to manufacturer's instructions.

    Techniques: Cell Surface Biotinylation Assay, Cell Culture, Labeling, Western Blot, Marker

    The C-terminal hTLR7 fragment localizes to the cell surface in the absence of the N-terminal hTLR7 fragment. ( A ) Colocalization of the HA-tagged C-terminal hTLR7 fragment (7C; red) with the early endosomal marker EEA1 (green) is reduced compared with the colocalization of full-length hTLR7 with EEA1 ( n ≥ 16). ( B ) Colocalization of the HA-tagged C-terminal hTLR7 fragment (7C; red) with CTxB (green). White arrows and white box indicate areas of colocalization ( n ≥ 9). In (A) and (B), THP-1 cells were fixed, permeabilized, blocked, and stained with the indicated Abs. Nuclear counterstain (DAPI) is shown in blue. Images were taken with a Zeiss inverted 780 confocal microscope. Quantification of colocalization was performed using Fiji. Li's coefficient was calculated to quantify the degree of colocalization. Data are represented as a box and whisker plot. Whiskers show minimum to maximum of all data. ( C ) Cell surface biotinylation of THP-1 cells expressing either HA-tagged full-length hTLR7 (TLR7) or the HA-tagged C-terminal hTLR7 fragment (7C), followed by immunoprecipitation using NeutrAvidin (Avidin) and Western blot analysis for HA tag. Control samples with no biotin are shown (-). ( D ) FACS staining with anti-FLAG Ab of THP-1 cells expressing the full-length hTLR7 and the C-terminal hTLR7 fragment with FLAG tag at the N terminus (F-7 and F-7C, respectively). Control staining with anti-FLAG Ab of THP-1 cells expressing the full-length hTLR7 and the C-terminal hTLR7 fragment without FLAG tag at the N terminus (7 and 7C) is also shown. Representative of at least three independent experiments. ** p

    Journal: The Journal of Immunology Author Choice

    Article Title: The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

    doi: 10.4049/jimmunol.1402703

    Figure Lengend Snippet: The C-terminal hTLR7 fragment localizes to the cell surface in the absence of the N-terminal hTLR7 fragment. ( A ) Colocalization of the HA-tagged C-terminal hTLR7 fragment (7C; red) with the early endosomal marker EEA1 (green) is reduced compared with the colocalization of full-length hTLR7 with EEA1 ( n ≥ 16). ( B ) Colocalization of the HA-tagged C-terminal hTLR7 fragment (7C; red) with CTxB (green). White arrows and white box indicate areas of colocalization ( n ≥ 9). In (A) and (B), THP-1 cells were fixed, permeabilized, blocked, and stained with the indicated Abs. Nuclear counterstain (DAPI) is shown in blue. Images were taken with a Zeiss inverted 780 confocal microscope. Quantification of colocalization was performed using Fiji. Li's coefficient was calculated to quantify the degree of colocalization. Data are represented as a box and whisker plot. Whiskers show minimum to maximum of all data. ( C ) Cell surface biotinylation of THP-1 cells expressing either HA-tagged full-length hTLR7 (TLR7) or the HA-tagged C-terminal hTLR7 fragment (7C), followed by immunoprecipitation using NeutrAvidin (Avidin) and Western blot analysis for HA tag. Control samples with no biotin are shown (-). ( D ) FACS staining with anti-FLAG Ab of THP-1 cells expressing the full-length hTLR7 and the C-terminal hTLR7 fragment with FLAG tag at the N terminus (F-7 and F-7C, respectively). Control staining with anti-FLAG Ab of THP-1 cells expressing the full-length hTLR7 and the C-terminal hTLR7 fragment without FLAG tag at the N terminus (7 and 7C) is also shown. Representative of at least three independent experiments. ** p

    Article Snippet: The cell surface biotinylation kit was from Pierce Thermo Scientific.

    Techniques: Marker, Staining, Microscopy, Whisker Assay, Expressing, Immunoprecipitation, Avidin-Biotin Assay, Western Blot, FACS, FLAG-tag

    Cell surface–localized chimeric hTLR7/4 is functionally inactive. ( A ) Western blot of THP-1 cells expressing either chimeric hTLR7/4 (TLR7/4) or full-length hTLR7 (TLR7) with anti-HA Ab. Control samples of THP-1 cells expressing GFP are shown. Black arrowheads indicate full-length hTLR7 and hTLR7/4; white arrowheads indicate the C-terminal hTLR7 fragment (in cells expressing full-length hTLR7) and the C-terminal fragment of hTLR7/4 (in cells expressing chimeric TLR7/4 constructs). ( B ) Cell surface biotinylation of THP-1 cells expressing HA-tagged chimeric hTLR7/4 (TLR7/4), followed by immunoprecipitation using NeutrAvidin (Avidin) and Western blot analysis for HA tag. Black arrowhead indicates full-length hTLR7/4; white arrowhead indicates the C-terminal fragment of hTLR7/4. Control samples with no biotin (-) and with biotinylated GFP-expressing THP-1 cells (GFP) are shown. ( C ) Colocalization of HA-tagged hTLR7/4 (TLR7/4) and full-length hTLR7 (TLR7) (green) with the membrane marker CTxB (red). Nuclear counterstain (DAPI) is shown in blue. Cells were fixed, permeabilized, and stained with Abs. Images were taken with a Zeiss inverted 780 confocal microscope. ( D ) Enhanced colocalization of TLR7/4 with the membrane marker CTxB ( n ≥ 7). Quantification of colocalization was performed using Fiji. Li's coefficient was calculated to quantify the degree of colocalization. Data are represented as a box and whisker plot. Whiskers show minimum to maximum of all data. ( E ) ELISA for IL-8 in THP-1 cells expressing the indicated constructs upon stimulation with the indicated concentrations of R837 for 24 h. Data are mean ± SD of triplicates. Representative of at least three independent experiments. *** p

    Journal: The Journal of Immunology Author Choice

    Article Title: The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

    doi: 10.4049/jimmunol.1402703

    Figure Lengend Snippet: Cell surface–localized chimeric hTLR7/4 is functionally inactive. ( A ) Western blot of THP-1 cells expressing either chimeric hTLR7/4 (TLR7/4) or full-length hTLR7 (TLR7) with anti-HA Ab. Control samples of THP-1 cells expressing GFP are shown. Black arrowheads indicate full-length hTLR7 and hTLR7/4; white arrowheads indicate the C-terminal hTLR7 fragment (in cells expressing full-length hTLR7) and the C-terminal fragment of hTLR7/4 (in cells expressing chimeric TLR7/4 constructs). ( B ) Cell surface biotinylation of THP-1 cells expressing HA-tagged chimeric hTLR7/4 (TLR7/4), followed by immunoprecipitation using NeutrAvidin (Avidin) and Western blot analysis for HA tag. Black arrowhead indicates full-length hTLR7/4; white arrowhead indicates the C-terminal fragment of hTLR7/4. Control samples with no biotin (-) and with biotinylated GFP-expressing THP-1 cells (GFP) are shown. ( C ) Colocalization of HA-tagged hTLR7/4 (TLR7/4) and full-length hTLR7 (TLR7) (green) with the membrane marker CTxB (red). Nuclear counterstain (DAPI) is shown in blue. Cells were fixed, permeabilized, and stained with Abs. Images were taken with a Zeiss inverted 780 confocal microscope. ( D ) Enhanced colocalization of TLR7/4 with the membrane marker CTxB ( n ≥ 7). Quantification of colocalization was performed using Fiji. Li's coefficient was calculated to quantify the degree of colocalization. Data are represented as a box and whisker plot. Whiskers show minimum to maximum of all data. ( E ) ELISA for IL-8 in THP-1 cells expressing the indicated constructs upon stimulation with the indicated concentrations of R837 for 24 h. Data are mean ± SD of triplicates. Representative of at least three independent experiments. *** p

    Article Snippet: The cell surface biotinylation kit was from Pierce Thermo Scientific.

    Techniques: Western Blot, Expressing, Construct, Immunoprecipitation, Avidin-Biotin Assay, Marker, Staining, Microscopy, Whisker Assay, Enzyme-linked Immunosorbent Assay

    GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. Biotinylated nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.

    Journal: bioRxiv

    Article Title: GRP78 and Integrins Play Different Roles in Host Cell Invasion During Mucormycosis

    doi: 10.1101/2020.04.29.069666

    Figure Lengend Snippet: GRP78 is a nasal epithelial cell receptor, while integrin α3β1 is an alveolar epithelial cell receptor during Mucorales interaction. Biotinylated nasal (A) or alveolar (B) epithelial cells were incubated with R. delemar germlings and unbound proteins were removed with repeated washing. Bound proteins were separated on SDS-PAGE, and identified by Western blotting using anti-biotin monoclonal antibody (Ab, top panel) and the identity of the proteins were confirmed to be GRP78 (78 kDa) for nasal (A) or integrin β1 (130 kDa) (B) by using anti-GRP78 or anti-Integrin α3β1 antibodies, respectively (bottom panels). Affinity purification of GRP78 (C) or integrin β1 (D), respectively, by other Mucorales. Anti-GRP78 and anti-integrin antibodies block R. delemar -mediated invasion and subsequent damage of nasal (E) and alveolar (F) epithelial cells when compared to isotype matched-IgG, respectively. Both antibodies had no effect on adherence of the fungus to host cells. Data in (E) and (F) are expressed as median ± interquartile range from 3 independent experiments. Different color codes are used to simplify the graph; purple, isotype IgG; green, anti-GRP78 Ab; and yellow, anti-integrin β1 Ab.

    Article Snippet: Thus, far-western blot analysis using recombinant human GRP78 and anti-GRP78 antibodies was done to identify R.delemar ligand.

    Techniques: Incubation, SDS Page, Western Blot, Affinity Purification, Blocking Assay

    Het α1 KO reduced β2 but not β3 subunit expression. A , we treated wild type brain slices with a membrane-impermeable biotinylation reagent and purified the biotinylated proteins with immobilized neutravidin. Equivalent fractions

    Journal: The Journal of Biological Chemistry

    Article Title: Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

    doi: 10.1074/jbc.M112.444372

    Figure Lengend Snippet: Het α1 KO reduced β2 but not β3 subunit expression. A , we treated wild type brain slices with a membrane-impermeable biotinylation reagent and purified the biotinylated proteins with immobilized neutravidin. Equivalent fractions

    Article Snippet: After incubation, the beads were pelleted by centrifugation and washed three times with RIPA buffer before the biotinylated protein was liberated using 60–80 μl of Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol.

    Techniques: Expressing, Purification

    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using biotinylated Protein A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.

    Journal: Protein Expression and Purification

    Article Title: Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli

    doi: 10.1016/j.pep.2015.07.008

    Figure Lengend Snippet: Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using biotinylated Protein A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.

    Article Snippet: The bound antibodies were then detected using biotinylated Protein A (Pierce) and biotinylated Protein G (Pierce) and streptavidin–phycoerythrin (Qiagen).

    Techniques: Recombinant, Infection, Luminex, Bead-based Assay, Binding Assay

    Relative EphA2 phosphorylation in the presence of different concentrations (50 μM, 25 μM, 12 μM, 6 μM) of compounds 1 (black), 2 (white) and 4 (sky blue). EphA2 phosphorylation was induced by treatment of PC3 cells with 0.25 μg/mL ephrin-A1-Fc. Cells were pretreated for 20 min with 1% DMSO or the indicated concentration of compounds and then stimulated for 20 min with ephrin-A1-Fc. Data are reported as a mean ± SEM of at least three independent experiments. One-way ANOVA followed by Dunnet’s post-test was performed to compare ephrin-A1-Fc + DMSO to all the other columns. * p

    Journal: Molecules

    Article Title: Synthesis and Structure-Activity Relationships of Amino Acid Conjugates of Cholanic Acid as Antagonists of the EphA2 Receptor

    doi: 10.3390/molecules181013043

    Figure Lengend Snippet: Relative EphA2 phosphorylation in the presence of different concentrations (50 μM, 25 μM, 12 μM, 6 μM) of compounds 1 (black), 2 (white) and 4 (sky blue). EphA2 phosphorylation was induced by treatment of PC3 cells with 0.25 μg/mL ephrin-A1-Fc. Cells were pretreated for 20 min with 1% DMSO or the indicated concentration of compounds and then stimulated for 20 min with ephrin-A1-Fc. Data are reported as a mean ± SEM of at least three independent experiments. One-way ANOVA followed by Dunnet’s post-test was performed to compare ephrin-A1-Fc + DMSO to all the other columns. * p

    Article Snippet: Compounds were added to the wells at proper concentration in 1% DMSO and incubated at 37 °C for 1 h. Biotinylated ephrin-A1-Fc (R & D Systems BT602) was added at 37 °C for 4 h at its KD in displacement assays.

    Techniques: Concentration Assay